Galectin-9 Is a High Affinity IgE-binding Lectin withAnti-allergic Effect by Blocking IgE-Antigen Complex FormationReceived for publication, June 18, 2009, and in revised form, August 28, 2009 Published, JBC Papers in Press, September 23, 2009, DOI 10.1074/jbc.M109.035196

Toshiro Niki‡1, Shoko Tsutsui‡, Shigeru Hirose‡, Sachiko Aradono‡, Yasushi Sugimoto‡, Keisuke Takeshita‡,Nozomu Nishi§, and Mitsuomi Hirashima‡¶

From the ‡Research Division, GalPharma Company, Ltd., FROM-Kagawa, 2217-16 Hayashi-cho, Takamatsu, Kagawa 761-0301and the §Life Science Research Center and ¶Departments of Immunology and Immunopathology, Faculty of Medicine,Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan

Galectin (Gal)-9was first described as an eosinophil chemoat-tractant. With the progress in research, Gal-9 has come to beknown as a versatile immunomodulator that is involved in vari-ous aspects of immune regulations, and the entire picture of thefunction still remains elusive. To uncover as-yet unknown activ-ity of Gal-9, we have been examining the effect of the protein invarious disease animal models. Here we show that Gal-9 atten-uated asthmatic reaction in guinea pigs and suppressed passive-cutaneous anaphylaxis in mice. These results indicate the mastcell stabilizing effect of Gal-9. In vitro studies of mast celldegranulation involving RBL-2H3 cells demonstrated thatGal-9 suppressed degranulation from the cells stimulated by IgEplus antigen and that the inhibitory effect was completely abro-gated in the presence of lactose, indicating lectin activity ofGal-9 is critical. We found that Gal-9 strongly and specificallybound IgE, which is a heavily glycosylated immunoglobulin, andthat the interaction prevented IgE-antigen complex formation,clarifying the mode of action of the anti-degranulation effect.Gal-9 is expressed by several mast cells including mouse mastcell line MC/9. The fact that immunological stimuli of MC/9cells augmented Gal-9 secretion from the cells implies thatGal-9 is an autocrine regulator of mast cell function to suppressexcessive degranulation. Collectively, these findings shed lighton anovel functionofGal-9 inmast cells and suggest a beneficialutility of Gal-9 for the treatment of allergic disorders includingasthma.

Galectin (Gal)2 is a family of lectins characterized by a con-served carbohydrate recognition domain exhibiting bindingspecificity to �-galactoside (1). One of the members, Gal-9, hastwo carbohydrate recognition domains tethered by a linkerpeptide and ismainly expressed in the epithelium of the gastro-intestinal tract and in immune cells (2–5). Gal-9, like othergalectins, does not have a signal sequence and is localized in thecytoplasm. However, it is secreted into the extracellular milieu

through poorly understood mechanisms and exerts biologicalfunctions by binding to the glycoproteins on the target cell sur-face via their carbohydrate chains.Two target glycoproteins of Gal-9 have been identified,

namely T-cell immunoglobulin and mucin containing-protein3 (TIM3) and CD44. TIM3 is expressed by several populationsof immune cells including terminally differentiated Th1 cellsand CD11b� monocytes. Gal-9 stimulates cell death of TIM3�

Th1 cells, leading to the termination of Th1-biased immunore-actions (6). On the other hand, Gal-9 promotes TNF� secretionfrom CD11b� TIM3� monocytes and enhances innate immu-nity (7). CD44 is an important adhesionmolecule for migratinglymphocytes and eosinophils. Gal-9 interactionwithCD44 pre-vents CD44 from binding to hyaluronic acid, which is a princi-pal ligand for CD44 and for providing a foothold for migratingcells; hence, attenuates accumulation of activated lymphocytesand eosinophils to the inflamed lesion (8).Other functions of Gal-9, such as in chemoattraction of

eosinophils, suppression of Th17 cell differentiation, or promo-tion of regulatory T-cell differentiation (9, 10) cannot beexplained either by TIM3 or CD44 with the limited knowledgewe have present, which leaves the possibility of other targetmolecules of Gal-9. Because lectin binding is more promiscu-ous than protein to protein interactions, it is possible that Gal-9has multiple target molecules to exert its various biologicalfunctions, as has been demonstrated in Gal-1 or Gal-3 (11–18).Mast cells play an important defense role in the frontline of

host immunity, whereas the excessive activation causes allergicor autoimmune disorders (19). Gal-9 expression has beenshown in human cord blood-derived mast cells (20), but thefunction of Gal-9 in mast cells has not been elucidated yet,although an effect of TIM3 activation in mast cells was demon-strated to augment Th2 cytokine production using a polyclonalanti-TIM3 antibody, which is described as agonistic to TIM3signaling (21).In this report we demonstrate anti-allergic activity of Gal-9

administration in animalmodels.We also show that Gal-9 is anIgE-binding protein and suppresses IgE-antigen complex for-mation,which underlines themode of action of the anti-allergiceffect of Gal-9.

EXPERIMENTAL PROCEDURES

Expression and Purification of Recombinant Gals—Theexpression and purification of recombinantGal-1,Gal-3,Gal-7,Gal-9, stable-formGal-9 (sGal-9), andmouse stable-formGal-9

1 To whom correspondence should be addressed. Tel.: 81-87-869-7417; Fax:81-87-869-7092; E-mail: niki.t@galpharma.com.

2 The abbreviations used are: Gal, galectin; sGal, stable-form galectin; TIM3,T-cell immunoglobulin and mucin domain containing-protein 3; PBS, Dul-becco’s phosphate-buffered saline without calcium and magnesium;PBS-T, PBS containing 0.05% Tween 20; BSA, bovine serum albumin; HSA,human serum albumin; OVA, ovalbumin; sRAW, specific airway resistance;DNP, 2,4-dinitrophenol; �-HEX, �-hexosaminidase; LTC4, leukotriene C4;FACS, fluorescence-activated cell sorter; ELISA, enzyme-linked immu-nosorbent assay.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 47, pp. 32344 –32352, November 20, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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(msGal-9) were described previously (22–24). Human Gal-4cDNA was amplified from first-strand cDNAs prepared fromthe poly(A)�RNA fraction of human placenta (OriGene Tech-nologies) using forward and reverse primers tagged with extra5� EcoRI (5�-cgtcctggattcccatggcctatgtccccgcaccg-3�) andXhoI(5�-cgaccgctcgagttagatctggacataggacaa-3�) sequences, respec-tively. The amplified cDNAwas digested with EcoRI and XhoI,and the resulting cDNA fragment was inserted into the EcoRI-XhoI site of pGEX-4T-2. The glutathione S-transferase fusionprotein of humanGal-4 was purified with a glutathione-Sepha-rose column (GE Healthcare). The purified protein wasdigested with thrombin (GEHealthcare), and then the glutathi-one S-transferase moiety was removed using a glutathione-Sepharose column. All the galectins were intensively dialyzedagainst Dulbecco’s phosphate-buffered saline without calciumand magnesium (PBS), and then endotoxin was eliminatedusing Cellufine ETclean L, a poly-�-lysine-conjugated resin(Chisso), followed by sterilization through a 0.2-�m filter. Allthe preparations were�95% pure on SDS-PAGEwith an endo-toxin level of �0.001 endotoxin units/�g (�0.0001 ng ofendotoxin/�g of protein) in limulus turbimetric kinetic assaywith a Toxnometer ET-2000 (Wako). The protein concentra-tion was determined using a bicinchoninic acid assay reagent(Pierce) with bovine serum albumin (BSA) as the standard. Themolar concentration was calculated using the following molec-ular weights: Gal-1, 14,700; Gal-3, 26,200; Gal-4, 35,900; Gal-7,15,100; Gal-9, 34,700; sGal-9, 33,100; smGal-9, 33,500.Animal Studies—All the protocols for animal studies were

approved by the animal care and biosafety committee ofKagawa University. To establish an asthmatic reaction, maleguinea pigs (Hartley, 6 weeks; Kudou) were sensitized bymeansof daily inhalation of 1% ovalbumin (OVA) for 10 min using anultrasonic nebulizer (Omron) for 8 days. After a 7-day interval,the animals were challenged bymeans of inhalation of 2%OVAfor 5 min to induce an airway resistance increase, and the spe-cific airway resistance (sRaw) was determined at the indicatedtime points using a respiratory analyzer Plumos-I (MIPS). Tosuppress the synthesis of endogenous glucocorticosteroids andfacilitate the late-phase asthmatic reaction, the animals wereinjected with 10 mg/kg metyrapone (Sigma) intravenously 24 hand 1 h before the challenge, whereas 10 mg/kg pyrilamine(Sigma) was injected intraperitoneally 30 min before the chal-lenge to avoid anaphylaxis shock. sGal-9 was administeredintraperitoneally (1 mg/body) 30 min before each inhalation ofOVA.The sRaw changewas calculated by subtracting the initialsRawvalue before the induction from the sRaws at the indicatedtime points and expressed as percentages of the initial sRawvalues. After the last sRawmeasurement, blood and bronchoal-veolar-lavage fluid were taken for analysis. Total and OVA-specific IgE and IgG1 in the serum were measured using ELISAkits from Dainippon-Sumitomo and Morinaga, respectively.Passive-cutaneous anaphylaxis was induced in female mice(BALB/c, 7 weeks, SLC) sensitized by intravenous administra-tion of mouse IgE anti-2,4-dinitrophenol (DNP) antibody(clone Spe7; Sigma) at 5 �g/mouse a day before the assay. Thefollowing day sGal-9 or ketotifen (Sigma) was administeredintraperitoneally at the indicated doses. After 30 min 30 �l of0.15% 2,4-dinitrofluorobenzene (Sigma) dissolved in acetone/

olive oil (4/1) was topically applied to the surface of the right earof each mouse, whereas the solvent was applied to the left earfor control. The level of ear edema formation was determinedby measuring the ear thickness using a calibrated thicknessgauge (Mitsutoyo) and expressed as the ear thickness changeaccording to the formula (R � L) � (R0 � L0), where R and Lrepresent the thickness of the right and left ears at the indicatedtime points, andR0 and L0 represent the thicknesses just beforethe 2,4-dinitrofluorobenzene challenge. Statistical analysis ofanimal studies were carried out using GraphPad softwarePrism, assessed by two-way analysis of variance, and consideredas significant at p� 0.01 (**) or p� 0.001 (***) compared with aPBS control.Cell Culture—Rat basophilic leukemia RBL-2H3 cells and

mouse mast cell line MC/9 were from RIKEN BioResourceCenter. Human mast cell line HMC-1 and human T-cell linesJurkat and Molt-4 were from ATCC. RBL-2H3 cells were cul-tured inminimal essential medium (Sigma) supplemented with10% fetal bovine serum, penicillin/streptomycin, and gluta-mine. MC/9 was maintained in Dulbecco’s modified Eagle’smedium (Sigma) with 10% fetal bovine serum, 0.05 mM 2-mer-captoethanol, interleukin-2 culture supplement (BD Bio-sciences), and penicillin/streptomycin. HMC-1 was cultured inIscove’s modified Dulbecco’s medium with 10% fetal bovineserum and penicillin/streptomycin. Jurkat and Molt-4 weremaintained in RPMI 1640mediumwith 10% fetal bovine serumand penicillin/streptomycin.Mediator Release from RBL-2H3 Cells—20,000 cells of RBL-

2H3 cells per well were seeded into 96-well plates a day beforethe assay and cultured at 37 °C in a humidified CO2 incubator.Each cell layer was washed once with assay buffer (Hanks’ bal-anced salt solution containing 20mMHepes and 1mg/ml BSA)and then stimulated by the addition of the optimized concen-trations of 0.1 �g/ml Spe7 and 0.016 �g/ml DNP-conjugatedhuman serum albumin (DNP-HSA; Sigma) or 1 �g/ml rat IgE(clone IR162; Serotec) and 0.89 �g/ml anti-rat IgE antibody(clone MARE1; Zymed Laboratories Inc.). Galectins wereadded at the indicated time points and concentrations. For�-hexosaminidase (�-HEX) assay, supernatants were collected60min after the addition of DNP-HSA orMARE1, whereas thecell layerswere lysedwith 0.1%TritonX-100 and collected. Theamount of �-HEX was determined by measuring the enzymeactivity as described (25) with minor modifications. Briefly, 10�l of each supernatant or cell lysate was mixed with 10 �l of 1.3mg/ml 4-nitrophenyl 2-acetamido-2-deoxy-�-D-glucopyrano-side (Sigma) dissolved in 100 mM citrate buffer (pH 4.5) andthen incubated at 37 °C for 60 min. The reaction was termi-nated by the addition of 160 �l of 0.2 M glycine (pH 10.4), andthe optical density was read at 405 nm. The amounts of hista-mine and leukotrieneC4 (LTC4)were determinedusing specificELISA kits fromSPI Bio andCayman, respectively, according tothe manufacturer’s instructions, except that 20 mM lactose wasadded to avoid interference with the ELISA by Gal-9 carriedover from the assay.Measurement of Intracellular Ca2� Mobilization—RBL-2H3

cells seeded into an optical-bottomed black 96-well plate(Nunc) at 20,000 cells/well a day before the assay were incu-bated in 1 �M Fluo-3 AM (Molecular Probes) dissolved in cal-

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cium assay buffer (Hanks’ balanced salt solution containing 20mMHepes, 1mg/ml BSA, 1mM probenecid) for 90min at roomtemperature. The cell layer was washed twice with the calciumassay buffer, and then the kinetics of Ca2� mobilization weremonitored by measuring the fluorescence change (excitation,485 nm; emission, 538 nm) using a microplate reader Fluoros-kan Ascent (Thermo Electron).Binding Assay—Human IgG1 and IgE from myeloid were

purchased from Calbiochem. IgG1 and IgEs (1 �g/ml) werecoated in 96-well plates (Nunc) by overnight incubation at 4 °C.After washing out the unbound antibodies with PBS containing0.05% Tween 20 (PBS-T), 10 mg/ml polyethylene glycol 4000(Merck) was added to the plates followed by incubation for 2 hat room temperature to reduce nonspecific binding. Biotiny-lated galectins prepared using an EZ-linkNHS-biotinylation kit(Pierce) were diluted to 0.1�Mwith 1mg/ml BSA in PBS-T andthen added to the plates followed by incubation for 1 h at roomtemperature. The average biotin incorporation rate was 1–2biotins per galectin according to the results obtained with abiotin quantitation kit (Pierce), and the biotinylation did notaffect the hemagglutination activity assessed with trypsin-treated fixed rabbit erythrocytes (26). After extensive washingwith PBS-T, galectins bound to the plates were detected by theaddition of streptavidin-conjugated horseradish peroxidase(Zymed Laboratories Inc.) and a colorimetric enzyme sub-strate, tetramethylbenzidine (KPL). The color developmentwas stopped in its linear range by the addition of 1 M phosphate,and the optical density was read at 450 nm with a microplatereader Benchmark plus (Bio-Rad). The values were compen-sated for the number of biotins per galectin and then expressedas relative binding activity in comparison to that of Gal-1. Sur-face plasmon resonance analysis was carried out using a Biacore3000 (Biacore). Galectins diluted to 1, 0.1, and 0.01 �Mwith therunning buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM

EDTA, and 0.005% surfactant P20) were applied to biotinylatedSpe7 immobilized on a SA-sensor chip (Biacore) at the flow rateof 10 �l/min at 20 °C. The kinetic constants of the interactionwere calculated using BIAevaluation 4.1 software. The effect ofgalectins on IgE-antigen interaction was examined on the sur-face of 96-well plates as well as on RBL-2H3 cell surface. Theassay in 96-well plates is carried out as follows. First, DNP-HSA(0.1 �g/ml) was coated in 96-well plates by overnight incuba-tion at 4 °C. After blocking the plates with 10 mg/ml polyethyl-ene glycol 4000, 0.1�g/ml biotinylated Spe7 dissolved in PBS-Tcontaining 1 mg/ml BSA was added followed by incubation for1 h at room temperature to allow the interaction between thebiotinylated Spe7 and DNP-HSA. Galectins of the indicatedconcentrations were added 10 min before the addition of thebiotinylated Spe7. The plates were washed extensively withPBS-T, and then the remaining antibodies were detected withstreptavidin-conjugated horseradish peroxidase and tetram-ethylbenzidine as described above. To examine dissociation ofIgE from RBL-2H3 cells, RBL-2H3 cell layers prepared in12-well plates by 1 day of culture from seeding at 80,000 cells/well were washed with FACS buffer (PBS containing 1 mg/mlBSA and 0.05% sodium azide), incubated with 0.1 �g/ml bio-tinylated Spe7 for 1 h on ice, and thenwashedwith FACS bufferto eliminate unbound Spe7. Then the cells were incubated with

1 �M sGal-9 for 1 h on ice, washed with FACS buffer, and thenincubated with 0.5 �g/ml streptavidin-conjugated phyco-erythrin (Biolegend) for 30 min to label the remaining biotiny-lated Spe7 on RBL-2H3 cells. After washing with FACS buffer,the cells were dissociated from the plates by pipetting in FACSbuffer containing 20mM lactose and 2mMEDTA, suspended in1 �g/ml propidium iodide in FACS buffer, and examined withFACSCalibur (BD Biosciences). To examine the effect of Gal-9on IgE-antigen interaction and IgE-anti-IgE antibody interac-tion on RBL-2H3 cells, the cell layers incubated with 0.1 �g/mlSpe7 or IR162 for 1 h on ice were washed with FACS buffer,incubated with 1 �M sGal-9 for 10 min on ice followed by theaddition of 0.016 �g/ml DNP-conjugated biotinylated BSA(Biosearch Technologies) or 0.89 �g/ml biotinylated-MARE1to the cell layers preincubated with Spe7 or IR162, respectively,and further incubated on ice for 30 min. The cell layers werewashed with FACS buffer and incubated with 0.5 �g/mlstreptavidin-conjugated phycoerythrin for 30 min. Then thecells were dissociated from the plates as described above,stained by propidium iodide, and examined with FACSCalibur.Periodate Oxidation of IgE—The protocol for antibody was

applied as described (27). Briefly, 1.5 �g of biotinylated Spe7was incubated in 20 mM sodium meta-periodate/PBS (or PBSfor control) at the concentration of 0.2 mg/ml for 25 h at 4 °C.The periodate reaction was terminated by the addition of thesame volume of 50% glycerol and overnight incubation at 4 °C.Gal-9 Measurement in the Cell Lines—MC/9, HMC-1, Jur-

kat, and Molt-4 cells were washed with PBS and suspended inlysis buffer composed of 50 mM Tris-HCl (pH 7.5), 100 mM

NaCl, 1% Triton X-100, and Complete Protease Inhibitor Mix-ture (Roche Applied Science) and lysed by sonication. Theamount of Gal-9 was measured with specific ELISA (28, 29),whereas the total protein amount was determined using bicin-choninic acid assay reagent with BSA as the standard.Gal-9 Release from Activated Mouse Mast Cell Line MC/9—

MC/9 cells were suspended in the culture medium containing20mM lactose, seeded intoU-bottomed 96-well plates at 20,000cells/well, and then stimulated by the addition of the indicatedconcentrations of Spe7 and DNP-HSA. Lactose was added tothe medium to diminish binding of Gal-9 to the cell surfaceglycans. The culture supernatants were collected to measurethe levels of LTC4 and Gal-9, whereas the remaining cells wereexamined by trypan blue excretion to determine living cellnumbers.

RESULTS

Gal-9 Suppresses Asthmatic Reaction and Passive CutaneousAnaphylaxis in Animal Models—It has been shown that Gal-9exerts eclectic functions in various types of immune cells,although entire picture of the immunomodulatory function ofthe protein remains elusive. As a part of our efforts to uncoveras-yet unknown functions ofGal-9 and explore possible utilitiesof the protein for the treatment of immunological disordersother than autoimmune diseases in which therapeutic effect ofGal-9 is well established in animal models (6, 10, 29, 30), weasked the effect of Gal-9 in asthma model in guinea pigs (Fig.1A). In this model animals sensitized with inhaled 1%OVAwaschallengedwith 2%OVA to induce increased airway resistance.

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Gal-9 was administered before each OVA inhalation fromthe sensitization phase. In this study we used sGal-9 in whichprotease-sensitive linker peptide of Gal-9 is eliminated tostabilize the protein for long storage and for in vivo applica-tion but without compromising the activity (24). sGal-9attenuated sRaw increase in both the immediate and latephases. The total and OVA-specific IgE and IgG1 levels in

serum determined after the last sRawmeasurement were notstatistically different between sGal-9 and control groups.The analysis of cells in bronchoalveolar-lavage fluid after thelast sRaw measurement showed significant decrease of totaland eosinophil numbers in sGal-9 treated group (total: PBS,8584 � 686/�l; sGal-9, 4679 � 974/�l; p � 0.01; eosinophil:PBS, 3514 � 427/�l; sGal-9, 1139 � 310/�l; p � 0.01),whereas lymphocytes showed a decreasing tendency bysGal-9 treatment without statistical significance (lympho-cyte: PBS, 312 � 29/�l; sGal-9, 160 � 71/�l).

The efficacy in the immediate asthmatic reaction impliesthat sGal-9 suppresses mast cell degranulation and promptedus to examine the effects of sGal-9 on passive cutaneous ana-phylaxis model, a simpler in vivo model to examine mast celldegranulation. In this model sGal-9 was administered intrap-eritoneally to mice sensitized with anti-DNP antibody (Spe7),and then the animals were challenged with the antigen on anear to induce ear edema. The administration of sGal-9 evidentlysuppressed the ear edema, 1 �g/mouse sGal-9 being equivalentto 20 �g/mouse or more of ketotifen (Fig. 1B). The administra-tion of sGal-9 affected neither the general appearance nor thebody weight increase of the animals throughout these two invivo studies.Gal-9 Suppresses Mediator Release from RBL-2H3 Cells—

The effect of Gal-9 on mast cell degranulation was furtherassessed in vitro using RBL-2H3 cells, a commonly used modelsystem to examinemast cell degranulation. In Fig. 2ARBL-2H3cells pretreated with various concentration of Gal-9 were stim-ulated by the addition of Spe7 plus the antigen DNP-HSA, andthen �-HEX release was measured. Gal-9 suppressed �-HEXrelease in a concentration-dependent manner; the efficacies ofsGal-9 (Fig. 2A, left) and the natural form Gal-9 (Fig. 2A, right)being indistinguishable, whereas the effect was completelyreversed in the presence of lactose. Table 1 summarizes theeffects of various galectins as IC50 values. Gal-3, -4, and -9exhibited inhibitory effects in the concentration range used,whereas Gal-1 andGal-7 failed. The potency rank order of theinhibition was sGal-9 (both human and mouse type) � Gal-9 � Gal-3 � Gal-4. The effect of Gal-9 was much greaterthan that of ketotifen, a histamine receptor antagonist with amast cell stabilizing effect, the IC50 values being 0.22 �M (7.3�g/ml) and 385 �M (119 �g/ml) for sGal-9 and ketotifen,respectively. The inhibitory effect of Gal-9 was not limited to�-HEX release, but histamine and LTC4 releases were alsosuppressed (Fig. 2, B and C).Next, we examined the effects of Gal-9 when it was added

before IgE, after IgE, or after IgE cross-linking (Fig. 2D). Theinhibitory effect was observed even after the addition of IgE,indicating that Gal-9 is effective on IgE-primed mast cells,which is consistent with the efficacy of sGal-9 in our passivecutaneous anaphylaxis in mice. A similar effect of Gal-9 wasobserved when the cells were stimulated by rat IgE (IR162) plusanti-rat IgE antibody (MARE1) (Fig. 2E).Gal-9 Suppresses the Intracellular Ca2� Mobilization—Be-

cause Ca2� influx is known to be a prerequisite step for thedegranulation of RBL-2H3 cells (31, 32), we monitored thechange in intracellular Ca2� concentration using a calciumindicator, Fluo-3. The addition of Gal-9 by itself stimulated a

FIGURE 1. In vivo effects of Gal-9 in asthma and allergy models. A, toinduce asthmatic reactions, guinea pigs were sensitized by inhaled 1% OVAfor 8 days and challenged with 2% OVA to induce asthmatic reactions, andthen sRaw was determined at the indicated time points. sGal-9 was adminis-trated intraperitoneally (1 mg/body) 30 min before the OVA treatments inboth the sensitization and challenge. The data are expressed by percentchanges in sRaw. The amount of total and OVA-specific IgE and IgG1 wasmeasured by ELISA using serum obtained after the last sRaw measurement.Data shown are the mean � S.E., n � 7 or 8; **, p � 0.01 compared with PBScontrol. B, effect on passive cutaneous anaphylaxis in mice. Ear edema wasinduced in mice by topical challenge with 2,4-dinitrofluorobenzene (DNFB)on the right ear of Spe7-sensitized mice. sGal-9 (left) or ketotifen (right) wasadministered intraperitoneally 30 min before the challenge. The thickness ofthe ears was measured and is expressed on the vertical axes as ear swelling(thickness of the right ear over the left; mean � S.E., n � 4 or 5). ***, p � 0.001compared with PBS control.

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Ca2� mobilization (Fig. 3A, upper), but nevertheless, this cal-cium signal did not stimulate degranulation as was shown inFig. 2. The aggregation of Fc�RI on the addition of IgE plusantigen stimulated a Ca2� mobilization (Fig. 3A,middle); how-ever, it was completely abolished in the presence of Gal-9 (Fig.3A, upper). These effects of Gal-9 were abrogated in the pres-

ence of lactose (Fig. 3A, lower). Wehypothesized that the Gal-9-depend-ent Ca2� mobilization might be thesignal that desensitizes cells as todegranulation. To test this hypothe-sis, lactosewas added to the cells aftertermination of the Gal-9-inducedCa2�mobilization followedby stimu-lationwith IgE plus antigen to deter-mine whether �-HEX is released(Fig. 3B). The addition of lactosereversed the inhibitory effect ofGal-9 on �-HEX release, indicatingthat Gal-9-dependent Ca2� mobili-zation has no direct connectionwith the anti-degranulation effect.Gal-9 Is an IgE-binding Protein

and Deprives IgE of Binding toAntigen—Because IgE is a highlyglycosylated protein, we examined ifGal-9 binds IgE by means of a solid-phase binding assay inwhich immo-bilized immunoglobulins on 96-wellplates were bound with biotiny-lated-galectins (Fig. 4A). The aver-age biotin incorporation rate was1–2 per each galectin, and the bioti-nylation did not affect the lectinactivity. Gal-9 exhibited prominentbinding to IgEs from human (mye-loid), mouse (Spe7), and rat (IR162),whereas the binding of Gal-1, -3, -4,and -7 to these IgEs was muchweaker. The IgE binding was simi-larly strong between the naturalform Gal-9 and the stable form,sGal-9. Gal-3 has been known as anIgE-binding protein, but the inter-action was much weaker than thatof Gal-9 even though our recombi-nant Gal-3 and the reported prepa-ration seems to be similar at least inhemagglutination activity, i.e. theconcentration for hemagglutinationof our preparation and the reportedone was 3 and 2 �g/ml, respectively,using the same referenced protocolinvolving trypsin-treated fixed rab-bit erythrocytes (33). These interac-tions were completely abrogated inthe presence of lactose. The bind-ing between IgE and galectins was

further studied by surface plasmon resonance analysis usingBiacore. The dissociation constants (Kd) obtained for immo-bilized Spe7 against sGal-9 and Gal-3 were 3.6 � 10�8 and2.4 � 10�6 M, respectively, which demonstrated the higheraffinity of the Spe7-Gal-9 interaction over that of Spe7-Gal-3in a different assay format.

FIGURE 2. Attenuation of mediator release from rat basophilic leukemia RBL-2H3 cells by Gal-9. A, shownis the effect on �-HEX release by sGal-9 and natural-form Gal-9. RBL-2H3 cells were incubated with the indi-cated concentration of Gal-9 for 10 min in the presence or absence of 20 mM lactose or sucrose. Then the cellswere sequentially added and incubated with Spe7 (0.1 �g/ml) for 30 min followed by DNP-HSA (0.016 �g/ml).The supernatants were collected 60 min after the stimulation for �-HEX assay, and the amount of the enzymereleased into the supernatants was expressed as the percentage of total �-HEX in the cells. w/o, without. B andC, shown is the effect on histamine and LTC4 release. RBL-2H3 cells were stimulated as above in the presence orabsence of 1 �M Gal-9 or sGal-9. The supernatants were collected 5 and 20 min after the stimulation forhistamine and LTC4 assays, respectively. D, shown is suppression of �-HEX release from IgE-sensitized RBL-2H3cells. Cells were stimulated as described in A, whereas sGal-9 was added 10 min before Spe7, 30 min after Spe7,or 30 min after the addition of DNP-HSA. E, the experimental procedure is basically the same to as in D, butRBL-2H3 cells were stimulated by rat IgE IR162 (1 �g/ml) and anti-rat IgE antibody MARE1 (0.89 �g/ml). All thedata are derived from a single representative experiment of more than two and are shown as the mean � S.D.(n � 3).

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Not only binding to IgE, but Gal-9 suppresses the interactionbetween IgE and antigen. When added to the interaction assaybetween Spe7 and immobilized DNP-HSA, Gal-9 suppressedIgE-antigen complex formation in a concentration-dependentmanner, whereas Gal-1 and Gal-3 were ineffective at 1 �M (Fig.4B, left). The interaction betweenGal-9 andDNP-HSA is at thebackground level in this assay. The effect of Gal-9 was alsocompletely abolished in the presence of lactose (Fig. 4B, right),which supports the idea that the target of Gal-9 is carbohydratechains on IgE. To further examine the relevance, Spe7 was sub-

jected to periodate oxidation to degrade carbohydrate chains.Periodate oxidation decreased the Spe7 amount in the reaction(Fig. 4C, left) probably because of the increased adsorption ofthe carbohydrate-less Spe7 to the plastic tube. In accordancewith the decreased amount, binding toDNP-HSA of periodate-treated Spe7 decreased comparedwith themock treatment, butthe binding was insensitive to Gal-9 (Fig. 4C, right). This obser-vation clarifies that carbohydrate chains on IgE are the primarytarget of Gal-9.We further confirmed the inhibitory effect by Gal-9 on RBL-

2H3 cell surface using the same amount of Spe7 and DNP-HSAfor the degranulation assay. sGal-9 showed a limited effect onripping pre-bound Spe7 out of RBL-2H3 cell surface (Fig. 4D)but suppressed DNP-HSA binding to Spe7 on the cell surface(Fig. 4E, left). We also examined if Gal-9 suppresses interactionbetween IR162 and MARE1 because Gal-9 suppressed degran-ulation fromRBL-2H3 cells stimulated by these antibodies (Fig.2E). Even in this case, Gal-9 significantly reduced MARE1attachment by about 40% (Fig. 4E, right).Induction of Gal-9 Release from Mast Cells—Gal-9 expres-

sion in mast cells is shown in human cord blood-derivedmast cells (20) and human mast cell line HMC-1 (34). Wehave repeatedly seen strong signals of Gal-9 out of mast cellsin immunohistochemical studies using mouse and human

FIGURE 3. Effect of Gal-9 on intracellular Ca2� mobilization. A, Fluo-3-AM-loaded RBL-2H3 cells were sequentially added with 1 �M sGal-9 (or PBS for control),0.1 �g/ml Spe7, and then 0.016 �g/ml DNP-HSA at 30-min intervals as indicated by the arrows. The assay was carried out in the absence (upper and middle) orpresence (lower) of 20 mM lactose. The fluorescence signal is expressed on the vertical axes as relative fluorescence change (F/F0) from the initial signal value(Mean � S.D., n � 3). B, no relations between Gal-9-dependent Ca2� mobilization and the anti-degranulation effect of Gal-9 are shown. The assay procedurewas basically the same as in Fig. 2A, except that 20 mM lactose or control buffer was added 10 min after the addition of 1 �M Gal-9, and then the cells werestimulated with Spe7 and DNP-HSA for �-HEX release (mean � S.D., n � 3). Representative data of twice reproduced are shown.

TABLE 1Effects of various galectins on RBL-2H3 degranulationThe same experimental procedure as in Fig. 2A was used to evaluate the effects ofeach galectin. The IC50 value was calculated as the concentration for 50% inhibitionfrom the inhibition curve using GraphPad software Prism and is presented as themean� S.E. from at least three independent experiments.When 50% inhibitionwasnot reached in the concentration range used, the IC50 values were assumed to bemore than the highest concentration. msGal-9, mouse stable Gal-9.

Reagent IC50 for �-HEX release

�M

Gal-1 �9Gal-3 2.39 � 0.46Gal-4 5.65 � 0.39Gal-7 �9Gal-9 0.24 � 0.04sGal-9 0.22 � 0.04smGal-9 0.22 � 0.01Ketotifen 385�66

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tissue sections (data not shown). ELISA analysis of Gal-9expression in cell lines demonstrated overwhelmingly highamounts of Gal-9 in mast cell lines over T-cell lines (Fig. 5A).Because mouse mast cell lineMC/9 retains Fc�RI-dependentsignaling for degranulation and proinflammatory mediatorrelease, we investigated if Gal-9 is released from the cellswith the activation. This cell line released Gal-9 constitu-

tively under the culture conditions we used. The applicationof Spe7 plus DNP-HSA to the culture stimulated the releaseof LTC4 at 30 min followed by the augmentation of Gal-9release at 48 h (Fig. 5B), with the highest release in thisexperiment of 7.4 ng/106 cells, which corresponds to about11% of total Gal-9 in the cells (63.6 � 1.8 ng/106 cells). Theviability of the cells monitored bymeans of trypan blue stain-

FIGURE 4. Binding of Gal-9 to IgE prevent IgE-antigen complex formation. A, human IgG1 from myeloid, human IgE from myeloid, mouse monoclonal IgESpe7, and rat monoclonal IgE IR162 were coated in 96-well plates and allowed to interact with biotinylated-galectins. Relative binding is expressed on thevertical axes in comparison with the binding of Gal-1 (mean � S.D., n � 3). B, prevention of IgE-antigen complex formation by Gal-9 in 96-well plates is shown.DNP-HSA-coated 96-well plates were incubated with 0.1 �g/ml biotinylated Spe7 for 60 min in the indicated concentration of galectins in the presence (right)or absence (left) of 20 mM lactose. The data are expressed by the percentage of Spe7 binding as the binding without galectins is 100% (mean � S.D., n � 3).C, no effect of Gal-9 on periodate-treated IgE is shown. Biotinylated Spe7 was subjected to periodate oxidation to disrupt carbohydrate chains. The sampleswere sieved by SDS-PAGE and silver-stained (left) and used for binding assay against DNP-HSA in the presence or absence of 1 �M sGal-9 and 20 mM lactose asindicated (right). The assay condition is basically the same as B. Periodate- and mock-treated Spe7 were used without compensating for the loss of proteinduring the treatment, and the binding of mock-treated Spe7 in the absence of sGal-9 and lactose was set as 100% (mean � S.D., n � 4). D, RBL-2H3 cellspre-bound with 0.1 �g/ml biotinylated Spe7 were incubated with 1 �M sGal-9 when indicated, and Spe7, which stayed on the cell surface, was detected bylabeling the cells with streptavidin-conjugated phycoerythrin and FACS analysis. The mean fluorescence values are shown in the bar chart (mean � S.D., n �3). Dark gray histogram, Spe7�sGal-9�; pale gray histogram, Spe7�sGal-9�; open histogram, Spe7�sGal-9�. E, RBL-2H3 cells pre-bound with 0.1 �g/ml Spe7 (left)or 1 �g/ml IR162 (right) were incubated with either 0.016 �g/ml DNP-conjugated biotinylated BSA or 0.89 �g/ml biotinylated-MARE1 in the presence orabsence of 1 �M sGal-9 as indicated. The binding of DNP-BSA or MARE1 to the cells was determined by labeling the cells with streptavidin-conjugatedphycoerythrin and FACS analysis. The mean fluorescent values are summarized in the bar chart (mean � S.D., n � 3). Dark gray histogram, Spe7�sGal-9�DNP�

or IR162�sGal-9�MARE1�; pale gray histogram, Spe7�sGal-9�DNP� or IR162�sGal-9�MARE1�; open histogram, Spe7�sGal-9�DNP� or IR162�sGal-9�MARE1�. Representative data of at least twice-reproduced experiments are shown.

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ing was �95% under all the conditions throughout theexperiment.

DISCUSSION

We demonstrated in a previous report that Gal-9 suppressesairway inflammation and Th2 cytokine release in a murinemodel of asthma by preventing CD44-hyaluronic acid interac-tion of activated lymphocytes and eosinophils (8). In the cur-rent study we examined Gal-9 administration in an asthmamodel in guinea pigs and observed a suppressing effect in bothimmediate- and late-phase asthmatic reactions. Gal-9 treat-ment attenuated eosinophil accumulation in the lung. This canbe explained by the suppression of CD44-dependent migrationof eosinophils by Gal-9, which is supposed to be the majormode of action in ameliorating the late-phase asthmatic reac-tion. The suppression at immediate-phase reaction, however, ispresumed as an effect of Gal-9 onmast cell degranulation or onimmediate reactions triggered by the degranulation, especiallyas Gal-9 administration did not decrease IgE level in the serum.The suppressive effect of Gal-9 on mast cell degranulation wasfurther supported in vivo using a murine passive cutaneousanaphylaxis model and confirmed by in vitro studies involvingRBL-2H3 cells.It is interesting to note that Gal-3 also suppressed degranu-

lation from RBL-2H3 cells at a 10-fold higher concentrationthan Gal-9, although Gal-3 was previously reported to stimu-late the degranulation from the cells (35). These differences canbe explained by the different assay formats, i.e. they added RBL-2H3 cells to Gal-3-coated plates, whereas we applied galectinsdirectly to the cell culture layers. BecauseGal-3 exhibits affinityto Fc�RI as well, it can aggregate Fc�RI to stimulate degranula-tion when immobilized at high density on a culture plate. How-ever, soluble Gal-3 cannot cross-link Fc�RI unless it forms astable dimer or oligomer, and it probably prevents the interac-tion between IgE and Fc�RI or between IgE and antigen.

We showed thatGal-9 prevented Spe7 frombinding toDNP-HSA, whereas Gal-1 and Gal-3 were ineffective in the concen-tration ranges used, which correlates well to the binding affinity

of these galectins as to IgE and thepotency rank order of the inhibitionin RBL-2H3 degranulation. Theseresults support the hypothesis thatthe anti-degranulation effect ofGal-9 is largely because of interfer-ence with IgE-antigen complex for-mation. Gal-9 suppressed the inter-action between rat IgE IR162 andanti-IgE antibody MARE1 on RBL-2H3 cells by 40%, although degran-ulation from the cells by these anti-bodies was completely suppressedby Gal-9. Probably, Gal-9 binding toIR162 is a steric hindrance forIR162-loaded Fc�RIs to be in closeproximity with each other byMARE1; hence, signal transductionand the following degranulation issuppressed. The exact binding site

of Gal-9 on IgEs and how the binding affects IgE structureremain to be investigated.Expression of TIM3 was reported in human cord blood-de-

rived mast cells (20) and mouse bone marrow-derived mastcells (21). Activation of TIM3 on the bone marrow-derivedmast cells by an agonistic polyclonal antibody was reported notto affect mast cell degranulation by immunological stimuli butto augment Th2 cytokine production and also to suppress mastcell apoptosis induced by interleukin-3 withdrawal (21). TIM3expression in RBL-2H3 cells was detected only by reverse tran-scription-PCR, its level being less than the detection limit onWestern blotting (data not shown), suggesting that the surfaceexpression of TIM3, if present at all, must be low. The applica-tion of Gal-9 to RBL-2H3 cells stimulated a Ca2� mobilization.This calcium signalmight come from theGal-9 interactionwithTIM3 in RBL-2H3 cells because Gal-9 binding to TIM3 wasreported to stimulate a Ca2� mobilization in Th1 cells (6). Nev-ertheless, this calcium signal does not seem to play a role in theobserved anti-degranulation effects of Gal-9 because the addi-tion of lactose after the calcium signal reversed the effect ofGal-9.Gal-9 expressionwas shown inT-cells, B-cells,macrophages,

endothelial cells, and fibroblasts in synovial tissue samples fromrheumatoid arthritis patients (29), and its release from T-cellswas demonstrated using Jurkat T-cells (34). These cells are aplausible source of Gal-9 at the site of inflammation in modu-lating immune responses. Because mast cells express signifi-cantly high amount of Gal-9 over T-cells, we investigated ifGal-9 is secreted from mast cells with degranulation stimuli.Activation of mouse mast cell line MC/9 by Spe7 plus DNP-HSA augmented Gal-9 release into the supernatant. This resultsuggests thatmast cells can be a source ofGal-9 and implies thatGal-9 acts as an autocrine regulator for mast cells to suppressexcessive degranulation.Mast cells release various proinflammatory mediators by

activation and trigger allergic diseases such as asthma, atopicdermatitis, allergic rhinitis, and allergic conjunctivitis (36–40).A large number of antagonists against those proinflammatory

FIGURE 5. Expression and release of Gal-9 from mast cells. A, shown is ELISA analysis of Gal-9 expression inT-cell lines (Jurkat and Molt-4) and mast cell lines (HMC-1 and MC/9). B, release of Gal-9 from mast cells by thedegranulation stimuli is shown. Mouse mast cell line MC/9 was incubated with the indicated concentrations ofSpe7 and DNP-HSA for 30 min or 48 h to measure the released LTC4 (left) or Gal-9 (right), respectively, usingspecific ELISA. After 48 h incubation, cell number was 5–30% higher in the presence of Spe7 and DNP-HSAcompared with the control. Therefore, the amount of Gal-9 in the culture supernatant was compensated by thecell number and expressed on the vertical axis as release of Gal-9 per 106 cells. Data are representative of at leasttwo experiments (mean � S.D., n � 3).

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mediators have been developed in an effort to cure uneasy andsometimes deadly symptoms of the disease. However, thoseantagonists offer limited benefit due to blocking only a part ofthe reactions triggered by many proinflammatory mediatorsreleased from activated mast cells. A humanized anti-IgE neu-tralizing antibody, Omalizumab, is a more radical approach forthe treatment of allergic diseases. This antibody is directedagainst the binding site of IgE for Fc�RI and prevents free IgEfrom attaching to mast cells, hence preventing IgE-mediatedproinflammatory mediator release as a whole (41). Gal-9 alsotargets IgE and attenuates the activation of mast cells triggeredby the cross-linking of Fc�RI. Gal-9 blocks the access of antigento the IgE, and the anti-degranulation effect was demonstratedeven after the attachment of IgE to Fc�RI on mast cells. Thisseems to be the beneficial characteristic of Gal-9 over the anti-IgE strategy.Mast cells of symptomatic patients are assumed tohave bound IgE, and Gal-9, but not Omalizumab, probablyexerts fast-acting efficacy under the conditions as well.We found that Gal-9 is an IgE-binding protein suppresses

IgE-antigen complex formation and, hence, prevents proinflam-matorymediator release frommast cells. Immunological stimula-tionaugments the secretionofGal-9 frommastcells, implying thatGal-9 is an autocrine regulator of mast cell activity. Gal-9 admin-istration suppresses immediate-phase asthmatic reaction inguineapigs,whichmanifests the anti-degranulationeffect ofGal-9in complex pathophysiological conditions where various poly-clonal IgEs are involved. Together with the previously reportedfunction of Gal-9 in suppressing CD44-dependentmigration ofinflammatory cells to the lesions, the current study would addmore value to Gal-9 as a potential candidate to allow the devel-opment of a novel protein drug for the treatment of asthma andallergic disorders.

Acknowledgments—We thank Keiko Furutani for technical assist-ance and Drs. Makoto Iwata (Tokushima Bunri University), ShigekiKatoh (Kagawa University), and Kimishige Ishizaka (La Jolla Insti-tute for Allergy and Immunology) for useful discussions.

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Anti-allergic Effect of Galectin-9

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Keisuke Takeshita, Nozomu Nishi and Mitsuomi HirashimaToshiro Niki, Shoko Tsutsui, Shigeru Hirose, Sachiko Aradono, Yasushi Sugimoto,

Blocking IgE-Antigen Complex FormationGalectin-9 Is a High Affinity IgE-binding Lectin with Anti-allergic Effect by

doi: 10.1074/jbc.M109.035196 originally published online September 23, 20092009, 284:32344-32352.J. Biol. Chem. 

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